Ultra-Sensitive Platform for Nucleic acid detection using a novel method, Scanning Digital polymerase chain reaction  (PCR)

ABSTRACT

A method for analyzing a target nucleic acid includes diluting nucleic acid targets and filling pico to femto-liter sized wells such that they contain a single target nucleic acid and one or more amplification reagents, amplifying the target in the individual wells, distinguishing wells containing amplicon from the target and amplicon from a variant of the target generated by polymerase error by using two differently labeled-hybridization probes, one hybridizing to the target and one hybridizing to a specific variant of the target; and analyzing target amplicons.

PRIORITY CLAIM

This application claims priority to U.S. Prov. Pat. Appl. No. 62/584,055filed Nov. 9, 2017, the contents of which are hereby incorporated byreference as if fully set forth herein.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

Preferred and alternative embodiments of the present invention aredescribed in detail below with reference to the following drawingfigures.

FIG. 1 illustrates a top view of a multi-layer, thin-film cassetteaccording to an embodiment of the invention;

FIG. 2 illustrates a cross-section of the high-efficiency single-celldroplet generating cassette of FIG. 1;

FIG. 3 illustrates a top view schematic of an active single-cell dropletgenerator according to an embodiment of the invention;

FIG. 4 illustrates a cross-section view of the active single-celldroplet generator of FIG. 3; and

FIGS. 5-11 illustrate a workflow and associated system according to anembodiment of the invention.

DETAILED DESCRIPTION

There is a clear need in biological related sciences to determine thepresence of low abundance nucleic acid sequences for gene expressionanalysis, mRNA analysis, vial load determination, and pathogendetection, among others. There is also a strong need in research forabsolute quantification of target nucleic acid sequences. Absolutequantification is possible by partitioning a quantitative PCR reactioninto 1 Os of thousands of individual femtoliter volumes, or wells. Eachwell contains a single target molecule (positive) or no target molecule(negative). Sample partitioning allows sensitive, specific detection ofsingle template molecules. The partitioning mitigates the effects oftarget competition, making digital PCR amplification less susceptible toinhibition and greatly improving the discriminatory capacity of assays.

The only other currently available technology to perform absolutequantification of nucleic acid sequences (also known as Digital PCR) isto split the PCR reaction materials into thousands of individualemulsion droplets. This process is expensive, complicated, andcumbersome to perform and requires three separate instruments, a dropletgenerator, a thermocycler (for PC), and a flow-based droplet analyzer.

Embodiments include a novel structure and method for performing digitalPCR using a low-cost, easy-to-use consumable and a combinedthermocycler/analyzer. A PCR supermix is deposited into a slidecontaining approximately 20,000 microwells with volumes on the order offemtoliters. Capping layers, such as plastic or glass, are then added toseal each well to form individual reaction chambers for subsequent PCR.

Once prepared, the femtoliter well chips may be placed into a fullyintegrated system (it can also be done using two separate systems, onefor PCR and one for the analysis) that performs the thermalcyclingrequired for PCR and then the analysis. The system may comprise a laserwith a beam focused to interrogate only one well at a time, at least onephotodetector for measuring the emitted fluorescence from eachindividual well, a laser steering assembly for scanning the laser overthe 20,000+ wells, and a programmable microcontroller. The system willalso likely need to be able to “align” the laser to the consumable sothat it “knows” where all of the wells are located. To do this, thepreferred embodiment will be a photodiode placed on the opposite side(likely under) the femtoliter well consumable.

Additionally, there is a clear need in biological related sciences todetermine the presence of low abundance nucleic acid sequences for geneexpression analysis, mRNA analysis, vial load determination, andpathogen detection, among others. There is also a strong need inresearch for absolute quantification of target nucleic acid sequences.Absolute quantification is possible by partitioning a quantitative PCRinto 10s of thousands of individual picoliter volumes, or wells. Eachwell contains a single target molecule (positive) or no target molecule(negative). Sample partitioning allows sensitive, specific detection ofsingle template molecules (i.e., the molecule of interest). Thepartitioning mitigates the effects of target competition, making digitalPCR amplification less susceptible to inhibition and greatly improvingthe discriminatory capacity of assays.

The only other currently available technology to perform absolutequantification of nucleic acid sequences (also known as Digital PCR) isto split the PCR reaction materials into thousands of individualemulsion droplets. This process is expensive, complicated, andcumbersome to perform and requires three separate instruments, a dropletgenerator, a thermocycler (for PC), and a flow-based droplet analyzer.

Embodiments include a structure and method for performing digital PCRusing a low-cost, easy-to-use consumable and a combinedthermocycler/analyzer. This is done by creating emulsion droplets usinga low-cost, thin-film technology with an optional method to measure thesize of the droplets, and to some extent, the contents of the droplets,just downstream of their production, all within the samestructure/cassette. By combining precision laser processing andmulti-layer laminates, an embodiment provides low-cost, high-efficiencyemulsion droplet generating cassettes (see FIG. 1 and FIG. 2). This 3-D,thin-film structure is unique and allows for the sample well to bepositioned directly over the droplet generating orifice. By locating thesample well directly over the droplet orifice, the suspended cells canbe allowed to settle to the bottom, via gravity, to greatly increase theresulting cell-in-droplet efficiency.

Preferably, within the same cassette, it is also possible to incorporatea current Coulter-style particle interrogation structure (which may bedescribed in one or more of U.S. Pat. Nos. 7,417,418, 7,515,268,7,520,164, 7,579,823, 8,171,778, 8,329,437, and 8,804,105). When theoptional Coulter orifice is added just downstream of the dropletfabricator, it is possible to measure the size of the particle usingdirect current (DC) and the contents of the droplet using simultaneousalternating current (AC). It is also feasible to use just DC or just ACcurrent instead of both simultaneously.

An embodiment includes a system that works with the above describedemulsion droplet generating cassette that will drive the dropletfabrication with Coulter orifice feedback to help control droplet sizeand (in some cases) single-cell encapsulation efficiency and/ordetermination. Control of droplet size, frequency, and efficiency can beaccomplished by varying the applied pneumatic pressure and/or vacuum tothe cassette. This system has the optional ability to perform thenecessary thermal cycling to PRC on the prepared droplets when desired.This is done by thermally cycling the Retrieval Sample Well (FIG. 1)prior to removal of the cassette from the system.

Alternate embodiments may include:

Instead of using the Coulter orifice for downstream QC and feedback, itis possible to run the sample through a flow cytometer immediately afterfabrication to determine approximate droplet size and contents. This maybe done with side-scatter (or forward light collection) andfluorescence.

The cassette could be simplified to have just the droplet orificestructure with no feedback.

FIG. 1 illustrates the multi-layer, thin-film cassette having thehigh-efficiency droplet fabrication orifice, the downstream Coulterorifice (for size and content determination), and the Retrieval SampleWell where optional PCR can be performed.

FIG. 2 illustrates a cross-section of the high-efficiency single-celldroplet generating cassette (from FIG. 1) showing the layers optionallyadvantageous to perform the emulsion droplet production and thedownstream Coulter orifice for electric impedance-based particleanalysis.

One of the major challenges in forming droplets containing single cells,is the inability to control when a droplet should be formed such that itcontains a desired cell. State of the art technologies use statisticalmodels and cell concentrations to drive the efficiency of cell/droplets.Currently available commercial systems claim efficiencies of up to only60%, and actual efficiencies can be much lower. Because the success ofdownstream single-cell sequencing operations depends on the success,efficiency, and purity, of correctly produced single-cell droplets,there is a strong market need for a highly efficient single-cell dropletgenerator that can produce droplets with desired cells (only) on demand.In addition, there are currently no commercially available dropletsystems with built-in quality control checks of any kind.

An embodiment includes the multilayer thin-film droplet generatordiscussed above in Disclosure A with the addition of an epi fluorescencesystem to detect the presence of the cells of interest as they approachthe droplet generating orifice. Also added is an electrical actuator(such as a piezoelectric actuator) capable of creating a transientpressure pulse to selectively force the desired cells through thedroplet generating orifice, thereby only creating droplets containingcells, and driving efficiency towards 100%.

Cell/droplet efficiency, as well as purity (i.e., only desired cells andnot debris) is absolutely critical as the success of downstream DNAsequencing operations relies heavily on both the percent efficiency anddroplet purity. The best commercially available single-cell dropletsystems have efficiencies approaching only 60%, and actual efficienciesare typically much lower. FIG. 3 shows the top-view of one possibleconfiguration for an active single cell droplet generator. FIG. 4 is across-section view of the droplet generator orifice showing the seventhin film layers. In the preferred embodiment, the fabricated dropletsflow through a downstream Coulter orifice to measure their size (DCcurrent) and contents (i.e., if a cell is inside) using AC current. TheCoulter data can be used in real-time to adjust the input variables(pressure transducer timing, input pressures and/or vacuums, andoptional DC voltage across the droplet orifice), thereby manipulatingthe size of the droplets and the resulting single-cell efficiency. Sucha system will become a powerful tool in the rapidly growing field ofsingle-cell genomics.

FIG. 3 illustrates a top view schematic of an active single-cell dropletgenerator. Epi fluorescence illumination is directed to the dropletgenerator orifice. When a cell of interest approaches the dropletgenerator orifice, the piezoelectric transducer is activated and thetransient pressure differential forces the cell downward, through thedroplet generator, thereby only producing droplets with the desiredcells.

FIG. 4 illustrates a cross-section view of the active single-celldroplet generator from FIG. 3 in a seven-layer thin-film cassette. Thecassette is fabricated with two outer translucent capping layers, threedouble-sided pressure sensitive adhesive layers with fluidic channel,and a central polyester layer. Cells in suspension flow across and overthe droplet orifice and into a waste reservoir. When a cell of interestapproaches the droplet generator orifice, the piezoelectric transduceris activated and the transient pressure differential forces the celldownward, through the droplet generator, thereby only producing dropletswith the desired cells. The resulting single-cell droplets flow into adownstream though an optional Coulter orifice where the size can bemeasured using direct current and the droplet constituents (i.e., if acell is present or not) can be determined using high-frequencyalternating current. Once the droplets pass the Coulter orifice theyflow to a reservoir where they are re-collected and used for subsequentDNA sequencing.

What is claimed is:
 1. A method for analyzing a target nucleic acid, themethod comprising the steps of: diluting nucleic acid targets andfilling pico to femto-liter sized wells such that they contain a singletarget nucleic acid and one or more amplification reagents; amplifyingthe target in the individual wells; distinguishing wells containingamplicon from the target and amplicon from a variant of the targetgenerated by polymerase error by using two differentlylabeled-hybridization probes, one hybridizing to the target and onehybridizing to a specific variant of the target; and analyzing targetamplicons.
 2. The method according to claim 1, wherein said amplifyingstep is a polymerase chain reaction and the one or more amplificationreagents includes one or more primer pairs.
 3. The method according toclaim 1, wherein said distinguishing step comprises scanning a laseracross each well individually.
 4. The method according to claim 1,wherein said analyzing step comprises detecting said amplicons byhybridization to detectably-labeled probes.
 5. The method according toclaim 1, wherein said analyzing step is conducted on amplicon from wellsthat were not distinguished in said distinguishing step.
 6. The methodaccording to claim 1, wherein said analyzing step comprises: determininga number of wells that contain only wild-type target; determining anumber of wells that contain only a variant of the target.
 7. The methodaccording to claim 6, wherein presence of wells containing only saidvariant is indicative of a disease.
 8. The method according to claim 7,wherein the disease is cancer.
 9. The method according to claim 6,wherein the variant is an allelic variant.
 10. The method according toclaim 9, wherein the allelic variant is a single nucleotidepolymorphism.